Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

A systematic-ecological approach to Baltic Sea ice studies of algae and protists

The seasonal occurrence of sea ice that annually covers almost half the Baltic Sea area provides a unique habitat for halo- and cold temperature-tolerant extremophiles. Baltic Sea ice biology has more than 100 years of tradition that began with the floristic observation of species by the early pioneers using light microscopic techniques that were the only thing available at the time. Since the discovery of life within sea ice, more technologies have become available for taxonomy. Electron microscopy and genetic evidence have been used to identify sea ice biota revealing increased numbers of taxa. Meanwhile ecologists have used light microscopic cell enumeration in addition to the chemical and physical properties of sea ice in attempts to explain the food web structure of sea ice and its functions. Thus, during the Baltic winter, the sea ice hosts more abundant and diverse microbial communities than the water column beneath it. These communities are typically dominated by autotrophic diatoms together with a diverse assortment of dinoflagellates, auto- and heterotrophic flagellates, ciliates, metazoan rotifers and bacteria, which are mostly responsible for the recycling of nutrients. This thesis comprises ecological and systematic studies. In addition to the results of the previous studies carried out on landfast ice, the data presented here provide new insight into the spatial distribution of pelagial sea ice, which has remained largely unexplored. The studies reveal spatial heterogeneity in the pelagial sea ice of the Gulf of Bothnia. There were mismatches in chlorophyll-a concentrations and in photosynthetic efficiencies of the communities studied. The temporal succession was followed and experimental studies performed investigating the community responses towards increased or decreased light in landfast ice in the Gulf of Finland. The systematic studies carried out with established dinoflagellate cultures revealed a new resting cyst belonging to common sea ice dinoflagellate, Scrippsiella hangoei (Schiller) Larsen 1995. The cyst can be used to explain the overwintering of this species during prolonged periods of darkness. The dissimilarities and similarities in the material isolated from the sea ice called for description of a new subspecies Heterocapsa arctica ssp. frigida. The cells obtained in the cultured material were unlike those of the previously described species, necessitating description of ssp. frigida. As a result of its own unique habitus, the subspecies had been noted by Finnish taxonomists during the past three decades and thus its annual occurrence and geographical distribution in the Baltic Sea. This illustrates how combining ecology and systematics increases our understanding of organisms.

Feeding on protists and particulates by the leptocephali of the worm eels Myrophis spp. (Teleostei: Anguilliformes: Ophichthidae), and the potential energy contribution of large aloricate protozoa

The food sources of the leptocephali of the teleostean superorder Elopomorpha have been controversial, yet observations on the leptocephali of the worm eels, Myrophis spp. (family Ophichthidae) collected in the northern Gulf of Mexico indicate active, not passive, feeding. Leptocephali had protists in their alimentary canals. Estimates of the physiological energetics of worm eels indicate that large aloricate protozoa including ciliates could provide substantial energy to these leptocephali toward the end of the premetamorphic and metamorphic stages, given the low energy requirements of metamorphosing leptocephali. Global ocean warming will likely force a shift in oceanic food webs; a shift away from large protozoa toward smaller protists is possible. Such a disruption of the oceanic food webs could further compromise the survival of leptocephali.

Comparison of phosphate uptake rates by the smallest plastidic and aplastidic protists in the North Atlantic subtropical gyre

The smallest phototrophic protists (<3 μm) are important primary producers in oligotrophic subtropical gyres – the Earth's largest ecosystems. In order to elucidate how these protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4–29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (<2 and 2–3 μm). Both groups of plastidic protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times (P<0.001) higher than those of bacterioplankton, the biomass-specific phosphate uptake rates of protists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition – predation on phosphorus-rich bacterioplankton cells.

Evaluation of the possible role of ants (Hymenoptera: Formicidae) as mechanical vectors of nematodes and protists

Os Nematoda e Protista podem ser transmitidos ao homem de diversas maneiras, mas pouca ênfase é dada para a transmissão mecânica por intermédio de formigas. Assim, esse trabalho procurou investigar a transmissão mecânica de ovos de Ascaris lumbricoides e cistos de Entamoeba coli pelos Formicidae. Através de experimentos com espécies mantidas em ninhos no laboratório (Tapinoma melanocephalum, Linepithema humile e Monomorium pharaonis) e com 17 espécies de formigas de uma área antropizada na região de Mogi as Cruzes (SP), foi possível constar que os ovos A. lumbricoides foram transportados por L. humile, tanto no campo (1 operária) como no laboratório (1 operária), por Camponotus rufipes (2), por Solenopsis saevissima (1) e por Acromyrmrex niger (1). em três operárias de C. rufipes foram encontrados cistos de E. coli. Apesar da baixa incidência de transporte, as três primeiras espécies pelo fato de viverem muito próximas ao ser humano, podem levar para dentro do ambiente domiciliar patógenos de Nematoda e Protista.